Journal: Cell Death & Disease

Article Title: The PP2A regulator IER5L supports prostate cancer progression

doi: 10.1038/s41419-024-06907-z

Figure Lengend Snippet: Volcano plot representation of the differentially expressed genes (DEGs) ( a ) and proteins ( b ) upon IER5L silencing by shScramble (shScr) or sh2 IER5L (shIER5L) transduction. The common targets between the RNAseq and proteomics’ analyses are highlighted. c Venn diagram summarizing the number of DEGs and the proteins affected by IER5L depletion in the RNAseq and proteomics experiments from ( a ) and ( b ). d Analysis of the expression of the indicated genes by qRT-PCR upon IER5L depletion by sh2 IER5L transduction in PC3 cells. The mRNA levels are normalized to GAPDH and shScr. The dotted line represents the normalized value of the shScr data. A one-sample t -test was applied for statistical analysis ( n = 3). Functional enrichment analysis of the DEGs upon IER5L silencing by KEGG ( e ) and Reactome ( f ). Left panels: Analysis of ARHGEF1 ( g ) and RCC2 ( h ) mRNA expression by qRT-PCR. The levels are normalized to GAPDH and non-target (siC) condition. The dotted line represents the normalized value of the siC data. A one-sample t -test was applied for statistical analysis ( n = 5 and n = 6 respectively). Middle panels: Analysis of foci formation upon ARHGEF1 ( g ) and RCC2 ( h ) depletion. The number of foci is shown. A paired t -test was applied for statistical analysis ( n = 5 and n = 4 respectively). Right panels: Representative images of the foci experiments are shown.

Article Snippet: Amplifications were run in a Viia7, QS5 or QS6 Real-Time PCR Systems (Applied Biosystems) using the following Taqman probes from Life Technologies: IER5L/Ier5l (Hs04186822_s1/ Mm07299543_s1), ARHGEF1 (Hs00180327_m1), FSCN1 (Hs00602051_mH), HK2 (Hs00606086_m1), PODXL (Hs01574644_m1), PTGES3 (Hs04187819_g1) and RCC2 (Hs00603046_m1).

Techniques: Transduction, Expressing, Quantitative RT-PCR, Functional Assay

Journal: Clinical Cancer Research

Article Title: Regulator of Chromosome Condensation 2 Identifies High-Risk Patients within Both Major Phenotypes of Colorectal Cancer

doi: 10.1158/1078-0432.ccr-14-3294

Figure Lengend Snippet: Figure 3. siRNA knockdown of RCC2 in HCT15 cells has functional consequences on cell growth and morphology. Depletion of RCC2 causes reduced proliferation and increased cell spreading of HCT15 cells (A and B). HCT15 cells were transfected either with negative control siRNA (mock) or with two different siRNA sequences against RCC2 (siRNA1 and siRNA2). The cells were assayed after 4 days of transfection. Representative Western immunoblots showing knockdown of RCC2 by the two different siRNAs with quantification bar charts (A). Verification of knockdown was obtained by three independent experiments for each of the two siRNAs. The protein expression was normalized relative to cells transfected with mock. B, quantification of cell proliferation. Cells were counted after four days of growth on a 35-mm Petri dish. Each bar represents the average cell number from three independent experiments. Error bars indicate the standard error of the mean. P value from Student t test for paired samples (two-tailed). Representative images of the morphology of mock-transfected cells (left) and RCC2- depleted cells (right; B). The morphologic changes following RCC2 depletion are accompanied by a rearranged actin filament network (C–E). HCT15 cells were transfected either with negative control siRNA (mock; C) or with siRNA sequences against RCC2 (D and E). After 4 days of transfection, the cells were fixed, stained with anti-RCC2 antibodies and phalloidin to label actin, and visualized using immunofluorescence confocal microscopy. An apparent increase in the number of large multinuclear cells was observed in cells with reduced RCC2 protein (asterisk). Scale bar represents 20 mm and applies to all images. Depletion of RCC2 causes cell-cycle arrest in the G2–M phase and increased levels of apoptosis (F– H). HCT15 cells were either left untreated or transfected with negative control siRNA (mock) or with siRNA sequences against RCC2 (siRNA1). F, visualizations of representative cell-cycle phase distributions for each treatment, as seen by flow cytometry. G, quantification bar charts for cell-cycle phase distributions. H, quantification bar charts for levels of apoptosis, as seen by TUNEL assay. Error bars indicate the SEM from at least three independent experiments with at least three biological replicates for each experimental variable. P values indicate significant differences in cell-cycle phase distributions between mock- and siRNA-transfected cells (two-tailed Student t test for paired samples).

Article Snippet: For Western blotting, rabbit polyclonal anti-RCC2 antibodies were obtained from Novus Biologicals (Cat. No. NB11040618), recognizing an N-terminal epitope between residue 1 and residue 50.

Techniques: Knockdown, Functional Assay, Transfection, Negative Control, Western Blot, Expressing, Two Tailed Test, Staining, Confocal Microscopy, Cytometry, TUNEL Assay

Journal: Journal of Oncology

Article Title: RCC2 Expression Stimulates ER-Positive Breast Tumorigenesis

doi: 10.1155/2020/5619462

Figure Lengend Snippet: Expression of RCC2 in breast tumor tissues. (a) Western blot analysis detected an immunosignal with a molecular weight of 56 kDa in ER + breast tumor tissues ( n = 13) and breast fibroadenoma ( n = 7). The analysis also detected an immunosignal at a molecular weight of 37 kDa using anti-GAPDH in these tissue samples. (b) RCC2 expression was normalized with GAPDH expression. (c) Immunohistochemistry detected significant RCC2 expression in breast tumor tissues. The analysis also detected the expression of RCC2 in endothelial cells of normal breast tissues. Original magnification: 200x. (d) Semiquantification of RCC2 expression in breast tumor and healthy breast tissues. The y -axis indicates the level of RCC2 expression. ∗∗∗ p < 0.001.

Article Snippet: siRNA oligonucleotides targeting the RCC2 gene were designed as the sequence 5′-AACAGCAAGCTGCTTACCGCA-3′ and synthesized by QIAGEN.

Techniques: Expressing, Western Blot, Molecular Weight, Immunohistochemistry

Journal: Journal of Oncology

Article Title: RCC2 Expression Stimulates ER-Positive Breast Tumorigenesis

doi: 10.1155/2020/5619462

Figure Lengend Snippet: Effect of RCC2 expression on MCF-7 cells. MCF-7 cells were cultured and transfected with anti-RCC2 siRNA (si-RCC2) or RCC2-expressing plasmids (O-RCC2). AllStars siRNA (si-AllStars) and RFP-expressing plasmids (O-RFP) were used as the respective controls. (a, b) Cell proliferation was measured using the CCK-8 assay. (c, d) Apoptosis was measured using flow cytometry. (e, f) Cell migration ability was measured using a wound healing assay. ∗ p < 0.05 and ∗∗ p < 0.01.

Article Snippet: siRNA oligonucleotides targeting the RCC2 gene were designed as the sequence 5′-AACAGCAAGCTGCTTACCGCA-3′ and synthesized by QIAGEN.

Techniques: Expressing, Cell Culture, Transfection, CCK-8 Assay, Flow Cytometry, Migration, Wound Healing Assay

Journal: Journal of Oncology

Article Title: RCC2 Expression Stimulates ER-Positive Breast Tumorigenesis

doi: 10.1155/2020/5619462

Figure Lengend Snippet: Assessment of the protein levels of IGF1 and TWIST1 in MCF-7 cells using Western blot analysis. MCF-7 cells were transfected with anti-RCC2 siRNA (si-RCC2) (a–c) or RCC2-expressing plasmids (O-RCC2) (d–f). Cells transfected with AllStars siRNA (si-AllStars) and RFP-expressing plasmids (O-RFP) were used as the corresponding negative controls. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: siRNA oligonucleotides targeting the RCC2 gene were designed as the sequence 5′-AACAGCAAGCTGCTTACCGCA-3′ and synthesized by QIAGEN.

Techniques: Western Blot, Transfection, Expressing

Journal: Journal of Oncology

Article Title: RCC2 Expression Stimulates ER-Positive Breast Tumorigenesis

doi: 10.1155/2020/5619462

Figure Lengend Snippet: Effect of RCC2 expression on tumor growth in a xenograft mouse model. (a) Mice ( n = 5) were injected with MCF-7 cells infected with lentivirus containing empty vector (blank). (b) Mice were injected with MCF-7 cells transfected with lentivirus containing anti-RCC2 shRNA (shRCC2) ( n = 5). (c) Tumor tissues were dissected at 28 days after cell injection. (d) The tumor volume is recorded every 4 days and depicted on a map. (e) The tumor weight is depicted on a map.

Article Snippet: siRNA oligonucleotides targeting the RCC2 gene were designed as the sequence 5′-AACAGCAAGCTGCTTACCGCA-3′ and synthesized by QIAGEN.

Techniques: Expressing, Injection, Infection, Plasmid Preparation, Transfection, shRNA

Journal: Journal of Oncology

Article Title: RCC2 Expression Stimulates ER-Positive Breast Tumorigenesis

doi: 10.1155/2020/5619462

Figure Lengend Snippet: Expression levels of IGF-1, RCC2, and TWIST1 in MCF-7 cells treated with estradiol-17 β (Ε 2 ) at a concentration of 10 −8 mol/L. Real-time PCR (a) and Western blot (b) were used to detect the transcriptional and translational levels of these targeted genes in MCF-7 cells, respectively. Cell culture in the presence of 0.1% ethanol was used as a control (con). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: siRNA oligonucleotides targeting the RCC2 gene were designed as the sequence 5′-AACAGCAAGCTGCTTACCGCA-3′ and synthesized by QIAGEN.

Techniques: Expressing, Concentration Assay, Real-time Polymerase Chain Reaction, Western Blot, Cell Culture, Control

Journal: Journal of Oncology

Article Title: RCC2 Expression Stimulates ER-Positive Breast Tumorigenesis

doi: 10.1155/2020/5619462

Figure Lengend Snippet: Effect of RCC2 expression on cell proliferation and apoptosis in MCF-7 cells cultured in the presence of estradiol-17 β at a concentration of 10 −8 mol/L. MCF-7 cells were transfected with anti-RCC2 siRNA (si-RCC2) or RCC2-expressing plasmids (O-RCC2). AllStars siRNA (si-AllStars) and RFP-expressing plasmids (O-RFP) were used as the corresponding controls. Untransfected cells were also used as a control. (a, b) Cell proliferation was measured using the CCK-8 assay. (c, d) Apoptosis was measured using flow cytometry. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: siRNA oligonucleotides targeting the RCC2 gene were designed as the sequence 5′-AACAGCAAGCTGCTTACCGCA-3′ and synthesized by QIAGEN.

Techniques: Expressing, Cell Culture, Concentration Assay, Transfection, Control, CCK-8 Assay, Flow Cytometry